PHYSIOLOGICAL RESEARCH • ISSN 0862-8408
(print)• ISSN 1802-9973
(online) 2018 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: physres@fgu.cas.cz, www.biomed.cas.cz/physiolres
RAPID COMMUNICATION
Dissecting the Role of Folr1 and Folh1 Genes in the Pathogenesis of Metabolic Syndrome in Spontaneously Hypertensive Rats
J. ŠILHAVÝ
1, J. KRIJT
2, J. SOKOLOVÁ
2, V. ZÍDEK
1, P. MLEJNEK
1, M. ŠIMÁKOVÁ
1, V. ŠKOP
3, J. TRNOVSKÁ
3, O. OLIYARNYK
3, I. MARKOVÁ
3, M. HÜTTL
3,
H. MALÍNSKÁ
3, L. KAZDOVÁ
3, F. LIŠKA
4, V. KOŽICH
2, M. PRAVENEC
11
Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic,
2Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic,
3Institute for Clinical and
Experimental Medicine, Prague, Czech Republic,
4Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic
Received April 18, 2018 Accepted June 26, 2018
Summary
Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.
Key words
Spontaneously hypertensive rat • Folr1 gene • Folh1 gene • Folate • Cysteine • Metabolic syndrome • Folate hydrolase • Glutamate carboxypeptidase
Corresponding author
M. Pravenec, Institute of Physiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
E-mail: pravenec@biomed.cas.cz
Introduction
Increased levels of plasma cysteine predispose to obesity and metabolic disturbances (Carter and Morton 2016, Elshorbagy et al. 2012). Since folate and B vitamins modulate metabolism of sulfur amino acids including cysteine, mild hypercysteinemia may be a secondary consequence of deficiencies of these vitamins (Shane 2010). Our recent linkage, congenic and transgenic rescue experiments in spontaneously hypertensive rats (SHR) revealed a mutated Folr1 (folate receptor 1) as a quantitative trait gene (QTG) when genetically determined reduced renal expression of Folr1 was associated with decreased renal folate reabsorption, lower folate levels, hypercysteinemia and metabolic disturbances (Pravenec et al. 2016). The Folr1 gene
(position on chromosome 1 at 166 Mbp) is closely linked to another candidate gene, Folh1 (folate hydrolase 1) which is also known as glutamate carboxypeptidase II (GCPII) (position on chromosome 1 at 150 Mbp). Dietary folates are composed of a mixture of monoglutamyl and polyglutamyl forms that are hydrolyzed to the monoglutamyl form prior to transport across the jejunal brush border membrane (Shane 2010). The Folh1 gene codes for an enzyme that is predominantly involved in the hydrolysis of dietary polyglutamyl folates by sequential cleaving terminal γ-linked glutamate residues from dietary polyglutamyl folates. However, it has been reported that in Sprague-Dawley rats Folh1 is not expressed in the small intestine and dietary folates are hydrolyzed by pancreatic γ-glutamyl hydrolase (Ggh) (Shafizadeh and Halsted 2007). In the current study, we tested the hypothesis that Folh1 allele of unrelated BN (Brown Norway) strain, which was derived from wild rats, is expressed in the small intestine and analyzed its effects on folate and sulfur amino acid levels and on parameters of lipid and glucose metabolism using SHR-1 congenic sublines. Our results provided evidence that both reduced renal folate reabsorption due to downregulated Folr1 renal expression and possibly reduced intestinal folate absorption due to downregulated or practically nonexistent intestinal Folh1 expression predispose the SHR to relative folate deficiency, hypercysteinemia and disturbances of glucose and lipid metabolism.
Materials and Methods
Animals
SHR/OlaIpcv rats (referred to as the SHR strain), SHR.BN-D1Rat272/Igf2 congenic strain (referred to as the SHR-1 congenic strain) (St Lezin et al. 1997), and SHR-1 sublines that harbor either Folr1 allele of BN origin and Folh1 allele of SHR origin (referred to as SHR.BN-Folr1 subline) or Folr1 allele of SHR origin and Folh1 allele of BN origin (referred to as SHR.BN- Folh1 subline), and the BXH/HXB recombinant inbred (RI) strains (Hübner et al. 2005) were housed in an air-conditioned animal facility and allowed free access to standard food (Altromin 1314 diet, Lage, Germany) and water. The SHR.BN-Folr1 and SHR.BN-Folh1 sublines were selected from (SHR x SHR-1)F2 rats (N=207) with the following primers that distinguished the SHR and BN Folr1 and Folh1 alleles. For the Folr1 gene promoter: F primer CCA CCA TAC CTT GGA GCA
GT, R primer CCC AAA TTC CAA ACA ACC TG; for the Folh1 gene intron: F primer ATG TGTGCG TGC GTA TTC AG, R primer TAG CTG CTG ACT TTG TTG G. The animals with appropriate recombinations were backcrossed to SHR strain and then heterozygotes were intercrossed and differential chromosome segments in both sublines were fixed and homozygous rats were used for phenotyping. Biochemical and metabolic phenotypes were assessed in 4 month old nonfasted male rats (N=8 per group). Tissues for biochemical analyses were collected from non-fasted rats between 9 and 10 AM. All experiments were performed in agreement with the Animal Protection Law of the Czech Republic and were approved by the Ethics Committee of the Institute of Physiology of the Czech Academy of Sciences, Prague.
Biochemical parameters
Folate levels in serum and urine were determined by the Folate III Assay Kit (Roche GmbH, Basel, Switzerland) (the coefficient of variation for the assays for folate is <5 %). Concentrations of total homocysteine, cysteine, glutathione (GSH), GSH precursor gamma-glutamylcysteine and GSH degradation product cysteineylglycine in plasma were determined by reversed-phase HPLC with fluorescent detection after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3- diazole-4-sulfonate. The reduction of disulfides and protein bound homocysteine and cysteine was performed with tris(2-carboxyethyl)phosphine as described previously (the coefficients of variation for the assays for homocysteine and cysteine are <3 %).
Blood glucose levels were measured by the glucose oxidase assay (Erba-Lachema, Brno, Czech Republic) using tail vein blood drawn into 5 % trichloroacetic acid and promptly centrifuged. NEFA levels were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Serum triglyceride concentrations were measured by standard enzymatic methods (Erba- Lachema, Brno, Czech Republic). Serum insulin concentrations were determined using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden).
Tissue triglyceride measurements
For determination of triglyceride concentrations in liver and soleus muscle, tissues were powdered under liquid N2 and extracted for 16 h in chloroform:methanol, after which 2 % KH2PO4 was added and the solution was
centrifuged. The organic phase was removed and evaporated under N2. The resulting pellet was dissolved in isopropyl alcohol and triglyceride content was determined by enzymatic assay (Erba-Lachema, Brno, Czech Republic).
Basal and insulin stimulated glycogen synthesis in skeletal muscle
For measurement of insulin stimulated incorporation of glucose into glycogen, diaphragmatic muscles were incubated for 2 h in 95 % O2 + 5 % CO2 in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 0.1 μCi/ml of 14C-U glucose, 5 mmol/l of unlabeled glucose, and 2.5 mg/ml of bovine serum albumin (Fraction V, Sigma, Czech Republic), with or without 250 μU/ml insulin. Glycogen was extracted and insulin stimulated incorporation of glucose into glycogen was determined.
Parameters of oxidative stress
Oxidative stress was measured according to the activities of anti-oxidative enzymes, concentrations of glutathione, the major physiological mechanism response to oxidative stress and concentrations of lipoperoxidation products. The activity of superoxide dismutase (SOD) was analyzed using the reaction of blocking nitrotetrazolium blue reduction and nitroformazan formation. Catalase (CAT) activity measurement was based on the ability of H2O2 and ammonium molybdate to combine to produce a color complex detected spectrophotometrically. The activity of seleno-dependent glutathione peroxidase (GSH-Px) was monitored by oxidation of gluthathione using Ellman’s reagent (0.01 М solution of 5,5'-dithiobis-2 nitrobenzoic acid). The concentration of reduced glutathione (GSH) was determined by the reaction of SH-groups using Ellman’s reagent. Glutathione reductase (GR) activity was measured by the decrease of absorbance at 340 nm using a millimolar extinction coefficient of 6220 M-1cm-1 for NADPH (using the Sigma assay kit). Lipoperoxidation products were assessed according to concentrations of thiobarbituric acid-reactive substances (TBARS) determined by assaying the reaction with thiobarbituric acid. Concentrations of conjugated dienes were analyzed by extraction in the media (heptan:isopropanol = 2:1) and measured spectrophotometric in heptan’s layer.
Folh1 sequencing
cDNA sequencing was performed on PCR
amplified products using an Applied Biosystems 3730xl DNA Analyzer and the BigDye Terminator v 3.1 Cycle sequencing kit (Applied Biosystems, Waltham, USA).
The PCR primers were: Folh1-6F 5'-TGC AGA CTC TCT GCA GTA GA-3' and Folh1-3007R 5'-GAA GAT AAC AAT GAA AAA TAG AAA-3'.
Western blotting
Tissues were homogenized in aqueous buffer (50 mM Tris pH 8, 120 mM NaCl and 0.5 % NP-40) supplemented by protease inhibitor coctail Complete (Roche, Basel, Switzerland) using TissueLyser (Qiagen, Hilden, Germany). Lysates were run on SDS-PAGE (10 % separating gel), proteins were blotted onto PVDF membranes Immobilon P (EMD Millipore Biosciences, Billerica, Massachusetts, USA). Membranes were incubated overnight at 4 °C with anti-GCPII (FOLH1) mouse monoclonal antibody (generous gift of Pavel Šácha and Jan Konvalinka, described by Rovenská et al. 2008) at final dilution 1:5,000. Secondary HRP-conjugated antibody was from GE Healthcare Bio-Sciences (Little Chalfont, UK), and signal was detected using ECL Prime chemiluminiscent detection kit (GE Healthcare Bio-Sciences, Pasching, Austria) and Hyperfilm ECL.
Gene expression
Real-time PCR analysis was used to determine expression levels of Folh1 gene in the intestine. Total RNA was isolated using standard methods and cDNA was prepared and analyzed by real-time PCR testing using QuantiTect SYBR Green reagents (Qiagen, Inc.
Valencia, USA) on an Opticon continuous fluorescence detector (MJ Research, Waltham, USA). For all real time PCR studies in which gene expression levels were compared between strains within a given tissue, the gene expression levels were normalized in relation to expression of an internal housekeeping gene encoding Ppia (peptidylprolyl isomerase A, also known as cyclophilin). The primer pair for detection Folh1 was:
Folh1-1526F TGA AGG CTT TGA AGG CAA AT and Folh1-1670R GCC TGA AGC AAT TCC AAG TC.
The primer pair for detection of cyclophilin was: F primer 5´-AGC ATA CAG GTC CTG GCA T; R primer 5´-TCA CCT TCC CAA AGA CCA C.
Statistical analysis of metabolic and physiologic studies and real time PCR
Summary results are expressed as means ± SEM.
Analysis of gene expression data was performed using the Relative Expression Software Tool (version REST 2009) that tests for significant differences by a randomization procedure (Pfaffl et al. 2002).
Bichemical and metabolic data were analyzed by One-way ANOVA with Holm Sidak testing for comparisons across three groups with subgroup comparisons made against the SHR strain as the control with adjustments for multiple comparisons.
Results
Linkage, sequence, and expression analyses
We have genotyped the RI strains using primers that distinguished SHR and BN Folh1 alleles and found that the strain distribution pattern of Folh1 alleles (at position 150 Mbp) is identical to that of the D1Arb15 marker (at position 154 Mbp) that was previously located at the peak of the QTL linkage for serum cysteine (Pravenec et al. 2016). Thus Folh1 gene itself is located at the peak of QTL linkage for plasma cysteine levels.
Sequence analysis of the SHR Folh1 allele when
compared to BN sequence revealed 2 amino acid substitutions (BN→SHR) p.Arg15Gly and p.Thr545Asn.
Compared to SHR rats that practically did not express Folh1, SHR-1 congenic rats with the Folh1 allele of the BN origin exhibited weak but significant expression of Folh1 in the small intestine (Fig. 1). Western blot analysis revealed low expression of FOLH1 protein in the intestines isolated from both SHR and SHR-1 congenic rats but there was no significant difference between the strains (data not shown).
Fig. 1. The expression of the Folh1 gene in the intestine. Real time PCR revealed significantly reduced expression of Folh1 mRNA in the intestine of the SHR when compared to the SHR-1 congenic strain. * denotes P<0.01.
Table 1. Metabolic phenotypes in SHR versus SHR.BN-Folr1 and SHR.BN-Folh1 congenic sublines.
Traits SHR SHR.BN-Folr1
subline SHR.BN-Folh1 subline
Serum folate (nmol/l) 37.8±1 37±1 36±2.1
Urinary excretion of folate (µg/min) 4.7E-04±9E-05 2.6E-04±2E-05* 3E-04±4E-05 Folate clearance (ml/kg/min) 0.047±0.007 0.023±0.002* 0.028±0.004*
Plasma total homocysteine (µmol/l) 4.3±0.4 3.4±0.5* 3.3±0.3*
Plasma total cysteine (µmol/l) 230±16 188±26* 194±23*
Plasma cysteinyl-glycine (µmol/l) 3.3±0.3 3±0.5 2.9±0.3*
Plasma glutathione (µmol/l) 58±12 61±9 57±4
Plasma γ-glutamylcysteine (µmol/l) 6.5±0.5 4.8±0.6* 5.1±1.1*
Body mass (g) 320±7 310±7 304±7
Epididymal fat mass (g/100g BW) 1.14±0.04 1±0.07* 0.96±0.33*
Serum glucose (mmol/l) 7.8±0.3 7±0.2* 6.7±0.1*
Serum insulin (nmol/l) 0.45±0.03 0.28±0.03* 0.27±0.03*
Serum triglycerides (mmol/l) 2.8±0.2 3.1±0.3 2.9±0.3
Serum NEFA (mmol/l) 0.77 ±0.08 0.9±0.07 1.11±0.05*
Liver triglycerides (µmol/g) 14.1±0.8 14.4±0.9 13.1±0.6
Muscle triglycerides (µmol/g) 3.6±0.9 5.5±0.6 3.7±0.3
Basal glycogenesis (nmol glucose/g/2 h) 80±11 100±11 75±20
Insulin stimulated glycogenesis (nmol glucose/g/2 h) 201±25 216±27 199±35
* denotes P<0.05 versus SHR control.
Table 2. Parameters of oxidative stress in the SHR and SHR.BN-Folr1 and SHR.BN-Folh1 congenic sublines.
Trait SHR SHR.BN-Folr1 SHR.BN-Folh1
Liver
SOD (U/mg) 0.161±0.016 0.129±0.008* 0.118±0.005*
GSH-Px (µMGSH/min/mg) 228±14 199±19 151±10*
GR (µM NADPH/min/mg) 108±6 122±10 111±8
CAT (µM H2O2/min/mg) 1409±67 1496±103 1518±69
GSH (mM/g) 24.7±1.7 32.7±2* 22.2±0.5
CD (nM/mg) 44.3±2.3 46.3±6 41.0±1.9
TBARS (nM/mg) 2.170±0.146 1.374±0.105** 1.707±0.113*
Kidney cortex
SOD (U/mg) 0.085±0.004 0.072±0.008 0.081±0.004
GSH-Px (µMGSH/min/mg) 135±10 131±9 111±3
GR (µM NADPH/min/mg) 37.8±2.5 41.8±2.8 36.9±3.4
CAT (µM H2O2/min/mg) 556±42 769±43* 614±26
GSH (mM/g) 17.0±1.1 22.5±0.5* 15.7±0.6
CD (nM/mg) 23.8±1.3 27.8±3.1 24.3±2
TBARS (nM/mg) 1.345±0.075 1.267±0.099 1.291±0.056
* and ** denote P<0.05 and P<0.005, respectively, when compared to SHR control.
Phenotyping of congenic sublines
Table 1 shows biochemical and metabolic phenotypes in SHR versus SHR.BN-Folr1 and SHR.BN- Folh1 congenic sublines. As can be seen, both sublines had similar levels of plasma folate as the SHR. On the other hand, both sublines showed significantly reduced renal folate clearance that was associated with reduced cysteine and homocysteine levels as well as reduced levels of cysteinyl-glycine and γ-glutamylcysteine when compared to the SHR. In addition, both sublines had significantly reduced plasma glucose and insulin which suggested increased sensitivity of tissues to insulin action, however, there was no difference in basal and insulin stimulated glycogenesis when both lines were compared to the SHR.
Furthermore, both sublines had significantly lower relative mass of epididymal fat which suggests reduced adiposity.
Parameters of oxidative stress
Table 2 shows that relative folate deficiency was associated with alterations in antioxidant enzyme activities especially in the liver and to the lesser extend in the kidney. The liver of both SHR.BN-Folr1 and SHR.BN-Folh1 sublines showed protection against oxidative tissue damage as reflected by reduced concentrations of lipoperoxidation products measured as conjugated dienes and TBARS.
Discussion
In the current study, we tested the separate effects of Folh1 and Folr1 gene variants on folate and sulfur amino acid metabolism and on parameters of glucose and lipid metabolism. We found that Folh1 allele of the BN origin, contrary to the SHR allele, is weakly but consistently expressed in the small intestine. Folh1 gene is located at the peak of QTL linkage for plasma cysteine levels and analysis of congenic sublines demonstrated that both Folr1 and Folh1 BN alleles were associated with reduced homocysteine and cysteine levels, and with amelioration of insulin resistance and decreased adiposity.
It has been reported that Folh1 mRNA and protein are not expressed in the intestine and pancreas isolated either from Sprague-Dawley outbred stock or LEW inbred strain (Shafizadeh and Halsted 2007, Rovenská et al. 2008) and it was suggested that dietary polyglutamyl folates in rats are hydrolyzed by pancreatic γ-GH (γ-glutamyl hydrolase) and instead of Folh1 (Shafizadeh and Halsted 2007). However, our results showed consistent intestinal mRNA expression of Folh1 in SHR-1 congenic strain. As can be seen in Figure 1, the expression of Folh1 in the SHR was negligible and it is possible that strains of Wistar origin, including LEW and
Sprague-Dawley as used in the former studies (Rovenská et al. 2008, Shafizadeh and Halsted 2007) and SHR used in the current study, do not express Folh1 in the intestine in significant amount while unrelated BN strain shows weak but consistent expression. Thus it is possible that Folh1 allele of the BN origin actually contributes to hydrolysis of polyglutamyl folates and absorption of folates from the intestine. On the other hand, Western blot analysis showed similar weak expression of FOLH1 protein in both SHR and SHR-1 strains.
Sequence analysis of the Folh1 gene revealed 2 amino acid substitutions, the p.R15G and the p.T545N which is more conserved: human, mouse and BN rats have threonine while the SHR has asparagine and the mutation is within the catalytic domain of the gene. It is not clear whether these mutations affect downregulation of Folh1 expression in the SHR.
Recently, we identified mutated Folr1 as a quantitative trait gene using transgenic rescue experiment (Pravenec et al. 2016). However, this transgenic experiment does not provide evidence against a possible role of Folh1 in folate metabolism and
disturbances of sulfur amino acids and lipid and glucose parameters. It is possible that transgenesis of Folr1
"rescued" both downregulated Folr1 and Folh1 SHR alleles. Phenotyping of SHR.BN-Folr1 and SHR.BN- Folh1 congenic sublines provided evidence that increased expression of both Folr1 and Folh1 alleles of the BN origin was associated with reduced cysteine and homocysteine levels, decreased adiposity and amelioration of insulin resistance. It can be concluded that the SHR is genetically predisposed to relative folate deficiency due to both reduced folate renal reabsorption and folate intestinal absorption, which is associated with hypercysteinemia, increased adiposity and insulin resistance.
Conflict of Interest
There is no conflict of interest.
Acknowledgements
This work was supported by grant 14-09283S from the Czech Science Foundation to VZ and VK.
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